Side-Chain Conformational Heterogeneity of Intermediates in the Escherichia coli Dihydrofolate Reductase Catalytic Cycle
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- 作者：Lisa M. Tuttle ; H. Jane Dyson ; Peter E. Wright
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：May 21, 2013
- 年：2013
- 卷：52
- 期：20
- 页码：3464-3477
- 全文大小：849K
- 年卷期：v.52,no.20(May 21, 2013)
- ISSN：1520-4995
文摘
Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the 蠂1 rotamer populations of the aromatic and 纬-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of 3JC纬CO and 3JC纬N coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of 3J data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The 3JC纬CO and 3JC纬N coupling constants are found to be extremely sensitive measures of side chain 蠂1 rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.